Coxsackie B viruses (CBV) interact with two receptors to initiate infection. All CBV initiate infection by attaching to the coxsackievirus and adenovirus receptor (CAR);many CBV isolates also bind to a second receptor, human decay accelerating factor (DAF). CBV, like other enteroviruses, are transmitted by the fecal-oral route in humans, and initiate infection by crossing the intestinal mucosa;however, CAR is not expressed on the apical surface of the polarized epithelial cells that line the intestinal lumen. In the first funding period of this grant, we have found that DAF is essential for infection of polarized cells. DAF permits virus to attach to the cell surface, but more importantly, it induces multiple intracellular signals that are required for virus uptake into the cell. We have found that additional virus-induced signals initiate non-apoptotic cell death by activating calcium-dependent proteases (calpains). Based on these observations, we propose a new series of experiments, focused on virus interaction with DAF, the role of DAF in infection by the enteral route, and the mechanisms and importance of calcium-mediated death signals. 1. We will define the site on the virus surface that permits attachment to DAF, and the site on the DAF molecule responsible for interaction with virus, using site-directed mutagenesis to test a model of the virus-DAF complex recently derived from cryo-em experiments. 2. We will determine whether DAF expression on the epithelial cell surface is important for in vivo infection by the enteral route, using transgenic mice expressing human DAF on intestinal epithelium. 3. We will define the signaling pathway that leads to necrotic cell death in cultured intestinal epithelium, determine whether calpain activation contributes to the pathogenesis of myocarditis and pancreatitis in infected animals, and test whether calpain inhibitors reduce the severity of disease. Relevance: Enteroviruses are the major cause of viral meningitis, and the second major cause of myocarditis in the US. Understanding how virus interacts with its intestinal receptor as it initiates infection, and understanding the mechanisms of virus-induced cell death, may provide new targets for therapeutic intervention.